Structural Analysis of Replication Protein A Recruitment of the DNA Damage Response Protein SMARCAL1

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• 作者：Michael D. Feldkamp ; Aaron C. Mason ; Brandt F. Eichman ; Walter J. Chazin
• 刊名：Biochemistry
• 出版年：2014
• 出版时间：May 13, 2014
• 年：2014
• 卷：53
• 期：18
• 页码：3052-3061
• 全文大小：562K
• 年卷期：v.53,no.18(May 13, 2014)
• ISSN：1520-4995

文摘

SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A-like1 (SMARCAL1) is a recently identified DNA damage response protein involved in remodeling stalled replication forks. The eukaryotic single-strand DNA binding protein replication protein A (RPA) recruits SMARCAL1 to stalled forks in vivo and facilitates regression of forks containing leading strand gaps. Both activities are mediated by a direct interaction between an RPA binding motif (RBM) at the N-terminus of SMARCAL1 and the C-terminal winged-helix domain of the RPA 32 kDa subunit (RPA32C). Here we report a biophysical and structural characterization of the SMARCAL1鈥揜PA interaction. Isothermal titration calorimetry and circular dichroism spectroscopy revealed that RPA32C binds SMARCAL1-RBM with a K_d of 2.5 渭M and induces a disorder-to-helix transition. The crystal structure of RPA32C was refined to 1.4 脜 resolution, and the SMARCAL1-RBM binding site was mapped on the structure on the basis of nuclear magnetic resonance chemical shift perturbations. Conservation of the interaction surface to other RBM-containing proteins allowed construction of a model for the RPA32C/SMARCAL1-RBM complex. The implications of our results are discussed with respect to the recruitment of SMARCAL1 and other DNA damage response and repair proteins to stalled replication forks.